Within our continued effort aimed at developing conjugate vaccines for infectious diseases from carbohydrate antigens we have three ongoing projects. Two are concerned with a vaccine for cholera and one with a vaccine for anthrax. Existing vaccines for these diseases are based on cellular material and, in addition to having undesirable side effects, they do not provide long-term immunity. Development of vaccines for these two diseases is important from both the point of view of public health and of the national interest. Development of a potent vaccine for cholera is important because of frequent involvement of our military in geographical areas where cholera is endemic. While anthrax does not constitute a major health problem in the civilized world, new concerns regarding anthrax have emerged because of potential use of some form of Bacillus anthracis, the etiological cause of anthrax, as a biological weapon. Our work towards a potent conjugate vaccine for cholera involves two approaches. In the first one, we synthesize oligosaccharides that mimic the structure of O-specific polysaccharide (O-PS) of Vibrio cholerae in the form suitable for conjugation, conjugate these antigens to suitable carriers, and serologically evaluate immunogenicity of the resulting neoglycoconjugates. In the second approach, we chemically modify polysaccharides isolated from bacterial pathogens to make them amenable for conjugation, conjugate the resulting synthons to suitable protein carriers, and use them as experimental vaccines. The approach towards a vaccine for anthrax is based on preparation of neoglycoconjugates from a suitable carrier and the chemically synthesized tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium. In the past, we have focused on improving diagnostic tools for the detection of presence of anthrax spores. Since preliminary work indicated that the anthrose-containing tetrasaccharide chain seemed to be highly specific for B. anthracis, during the period associated with this report we focused on testing the possibility of altering immunogenicity of the protective antigen (PA), which is protein in nature, by coupling it with the tetrasaccharide moiety from the BclA protein. Preliminary immunization studies suggested that this tetrasaccharide construct might enhance the immune response generated by PA. This was indicated by approximately 20% increase of protection of mice challenged with Ames spores. Due to lack of funds on the side of our collaborators, further work on optimizing the conjugates had to be temporarily discontinued. Very recently, the pharmaceutical company Novartis Vaccines in Siena, Italy, has shown interest in exploring possibilities to turn the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium synthesized in our laboratory into a vaccine for anthrax. Also, Novartis Vaccines is interested in looking at some immunochemical fundamentals concerning anthrax using synthetic fragments of the tetrasaccharide side chain of the major glycoprotein of Bacillus anthracis exosporium which we synthesized in the past. Based on the Material Transfer Agreement we have established with Novartis Vaccines, we have provided the aforementioned materials to our Italian collaborators. Work towards those goal is ongoing, being conducted by the Novartis team, headed by Dr. Roberto Adamo. In the cholera project, while immunization studies are still ongoing, we are continuing with the work on localization of site of conjugation of synthetic antigens on the carrier protein by mass spectrometry. Also, we are expanding the work based on our discovery we made in the past that bacterial O-SPcore antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. We have optimized our laboratory-scale protocol to make it useful for preparation of large batches of experimental vaccine from O-PScore of Vibrio cholerae O1 and a recombinant tetanus toxin fragment, and a protocol allowing large-scale preparation of cholera vaccine under conditions of cGMPs is under development by an outside of NIH contractor.